Nucleic acid sequence amplification technology has a wide application in bioscience, genetic engineering, and medical science for research and development and diagnostic purposes. In particular, the nucleic acid sequence amplification technology using PCR (hereafter referred to as “PCR amplification technology”) has been most widely utilized. Details of the PCR amplification technology have been disclosed in U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188.
Various apparatuses and methods incorporating automated PCR amplification processes have been developed and used for fast and efficient amplification of a variety of genetic samples. The basic working principle of such technology is as follows.
In the commercialized PCR amplification technology, a sample is prepared to contain a template DNA to be amplified, a pair of oligonucleotide primers complementary to a specific sequence of each single strand of the template DNA, a thermostable DNA polymerase, and deoxyribonucleotide triphosphates (dNTP). A specific portion of the nucleic acid sequence of the template DNA is then amplified by repeating a temperature cycle that sequentially changes the temperature of the sample. Typically, the temperature cycle consists of three or two temperature steps, and the amplification processes during the temperature cycle occur in the following manner. The first step is the denaturation step in which the sample is heated to a high temperature and double stranded DNA molecules become separated into single stranded DNA molecules. The second step is the annealing step in which the sample is cooled to a low temperature and the single stranded DNA molecules formed in the first step bind to the primers, forming partially double stranded DNA-primer complexes. The last step is the polymerization step in which the sample is maintained at a suitable temperature and the primers in the DNA-primer complexes are extended by the action of the DNA polymerase, generating new single stranded DNA molecules that are complementary to each of the template DNA strands. The target nucleic acid sequences as selected by the sequences of the two primers are replicated during each cycle consisting of the above three steps. Typically, several millions or higher number of copies of the target nucleic acid sequences can be produced by repeating the temperature cycles for about 20 to 40 times.
The temperature of the denaturation step is typically 90˜94° C. The temperature of the annealing step is controlled appropriately according to the melting temperatures (Tm) of the primers used, and it typically ranges from 40 to 60° C. It is typical to set the temperature of the polymerization step to 72° C. and use a three-step temperature cycle, since the most frequently used Taq DNA polymerase (a thermostable DNA polymerase extracted from Thermus aquaticus) has its optimal activity at that temperature. A two-step temperature cycle in which the polymerization temperature is set to the same as the annealing temperature, can also be used since the Taq DNA polymerase has its polymerase activity in a broad temperature range.
The prior nucleic acid sequence amplification methods have a number of drawbacks as they operate to change the temperature of the whole sample including DNA polymerase according to the three- or two-step temperature cycle.
Firstly, since DNA polymerase is included in the sample in the prior nucleic acid amplification methods, it is not simple to remove the DNA polymerase for purification of the sample after the amplification reaction, and also difficult to reuse the used enzyme.
Secondly, the prior nucleic acid sequence amplification methods can only use thermostable DNA polymerases such as Taq DNA polymerase. This is because the prior apparatuses have the process of heating the whole sample to a high temperature.
Thirdly, it is difficult to incorporate the prior nucleic acid sequence amplification method into a complex device such as Lab-on-a-chip, a miniaturized device that can perform multiple reactions and processes within a chip either simultaneously or sequentially. The prior nucleic acid sequence amplification method is disadvantageous for miniaturization since it requires processes of changing the temperature of the whole sample, thereby having a complex design and processes. Moreover, it is difficult to incorporate the prior method into a complex device in which rapid temperature change is not desirable.
Among various possibilities, a method useful for resolving the problems described above is one using immobilized DNA polymerase. By the term “immobilized enzyme” is meant an enzyme that is physically or chemically bound to a supporting material with its enzyme activity preserved. There are generally a number of advantages of using immobilized enzymes. Firstly, the sample purification process can be simplified since the enzyme can be readily separated and recovered from the reaction solution by removing the supporting material to which the enzyme is immobilized. Secondly, the cost can be reduced since the recovered immobilized enzyme can be reused. Thirdly, the efficiency of the reaction processes can be improved since multiple reaction processes comprising the enzyme reaction(s) can be simplified. In addition, immobilization of the enzyme may result in incidental effects such as improvement of the physical stability of the enzyme or change in the reaction conditions of the enzyme, which in turn may improve the applicability of the enzyme. Therefore, one can expect that the problems associated with the prior nucleic acid amplification methods described above can possibly be resolved by using immobilized DNA polymerase.
However, no method using an immobilized enzyme has been known yet in the prior art to solve the above problems. This is mainly due to two reasons: difficulty in preserving the activity of the immobilized enzyme and development of processes suitable for using the immobilized enzyme. Firstly, various methods has been reported for immobilization of enzymes, but no method has been reported yet for preparation of the immobilized DNA polymerase having high enough activity to produce detectable amount of reaction products in the PCR process. Therefore, in order to realize a nucleic acid amplification method and an apparatus thereof, a method should be developed first to prepare an immobilized DNA polymerase with its activity highly preserved. Secondly, even if an immobilized DNA polymerase preserving a high activity can be used, the prior methods of the temperature cycle type have limitations. For instance, non-thermostable DNA polymerases cannot be used in the prior temperature cycle type methods because the prior methods require a step of heating the whole sample to a high temperature. Moreover, in the prior methods, the polymerization reaction by DNA polymerase can occur only in one temperature step, namely the polymerization step. That is, the polymerization reaction can only occur for a partial period of the total reaction time. The temporal efficiency of the prior methods is thus limited by the speed of changing the temperature during the temperature cycle. Therefore, it is necessary to develop a new nucleic acid sequence amplification method and an apparatus thereof that are not of the prior temperature cycle type in which temperature of the whole sample is changed sequentially, but that are of a different type in which the advantages of using the immobilized DNA polymerase can be incorporated.